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PubMed1992

Matrix vesicles are enriched in metalloproteinases that degrade proteoglycans.

This study examined the presence of extracellular matrix processing enzymes in matrix vesicles produced by rat costochondral resting zone and growth zone chondrocytes in culture. Optimum procedures for the extraction of each enzyme activity were determined. Enzyme activity associated with chondrocyte plasma membrane microsomes was used for comparison. There was a differential distribution of the enzyme activities related to the cartilage zone from which the cells were isolated. Acid and neutral metalloproteinase (TIMP), plasminogen activator, and beta-glucuronidase were highest in the growth zone chondrocyte (GC) membrane fractions when compared with matrix vesicles and plasma membranes isolated from resting zone chondrocyte (RC) cultures. There was a threefold enrichment of total and active acid metalloproteinase in GC matrix vesicles, whereas no enrichment in enzyme activity was observed in RC matrix vesicles. Total and active neutral metalloproteinase were similarly enriched twofold in GC matrix vesicles. TIMP, plasminogen activator, and beta-glucuronidase activities were highest in the plasma membranes of both cell types. No collagenase, lysozyme, or hyaluronidase activity was found in any of the membrane fractions. The data indicate that matrix vesicles are selectively enriched in enzymes which degrade proteoglycans. The highest concentrations of these enzymes are found in matrix vesicles produced by growth zone chondrocytes, suggesting that this may be a mechanism by which the more differentiated cell modulates the matrix for calcification.

Animals↗

PubMed1997

Carbohydrate-mediated regulation of interaction of Vibrio cholerae hemolysin with erythrocyte and phospholipid vesicle.

Vibrio cholerae hemolysin is an extracellular pore-forming monomeric protein with a native molecular weight of about 60,000. In this study, we showed that the hemolysin interacted with immobilized phospholipids and cholesterol and formed oligomers in vesicles constituted from phospholipids alone with a stoichiometry identical to those produced in rabbit erythrocyte membrane. However, the hemolysin bound to glycoproteins with terminal beta1-galactosyl residues and an association constant of 9.4 x 10(7) M(-1) was estimated for the hemolysin-asialofetuin complex by solid phase binding assay. Oligomerization of the hemolysin in lipid bilayer converted the sugar-binding monomer to a lectin with strong carbohydrate-dependent hemagglutinating activity accompanied by inactivation of hemolytic activity and loss in ability to interact with phospholipids. There was no evidence for erythrocyte surface carbohydrates playing an essential role in interaction of the hemolysin with the cell. However, specific glycoproteins inhibited hemolysis of rabbit erythrocytes as well as interaction of the hemolysin with phospholipid. The results suggest (i) V. cholerae hemolysin is a monomer with distinct domains associated with specific binding to carbohydrates and interaction with lipids, (ii) the pore-forming property depends solely on the protein-lipid interaction with no evidence for involvement of sugars, and (iii) specific sugars can down-regulate the ability of the hemolysin to form pores in lipid bilayers.

Animals↗

PubMed1992

Matrix vesicles contain metalloproteinases that degrade proteoglycans.

This study explored whether extracellular matrix processing enzymes are present in matrix vesicles produced by rat costochondral resting zone and growth zone chondrocytes in culture. It was found that there was a differential distribution of enzyme activities related to the cartilage zone from which the cells were isolated. There was a 3-fold enrichment of total and active acid metalloproteinase in growth zone chondrocyte (GC) matrix vesicles whereas no enrichment in enzyme activity was observed in resting zone chondrocyte (RC) matrix vesicles. Total and active neutral metalloproteinase were similarly enriched 2-fold in GC matrix vesicles. TIMP, plasminogen activator and beta-glucuronidase activities were highest in the plasma membranes of both cell types. No collagenase, lysozyme, or hyaluronidase activity was found. The data indicate that matrix vesicles are selectively enriched in enzymes that degrade proteoglycans. The highest concentrations of these enzymes are found in matrix vesicles produced by growth zone chondrocytes, suggesting that this may be a mechanism by which the more differentiated cell modulates the matrix for calcification.

Animals↗

PubMed1983

Heparan sulfate proteoglycans from mouse mammary epithelial cells. A putative membrane proteoglycan associates quantitatively with lipid vesicles.

Mouse mammary epithelial (NMuMG) cells produce both cellular and extracellular heparan sulfate-rich proteoglycans. A cellular proteoglycan, but no extracellular proteoglycans, associates quantitatively and vectorially with lipid vesicles, as assessed by column chromatography and centrifugation. This lipophilic cellular proteoglycan is extracted as an aggregate when cells are treated with 4 M guanidine HCl, but is extracted as a single component in the presence of detergent, suggesting that it aggregates with cellular lipid. An association with lipid is confirmed by intercalation of the proteoglycan into the bilayer of lipid vesicles. Formation of lipid vesicles in the presence of the proteoglycan causes the proteoglycan to have the chromatographic and sedimentation behavior of the vesicles while destruction of the vesicles with detergent nullifies this effect. The proteoglycan is intercalated nullifies this effect. The proteoglycan is intercalated into the vesicles with its glycosaminoglycan-containing domain exposed to the exterior since mild trypsin treatment quantitatively removes this portion of the proteoglycan from the vesicle. After cleavage from the vesicle, the released proteoglycan chromatographs with an apparent molecular size similar to that of the whole proteoglycan, but no longer aggregates with lipid. Thus, trypsin removes a lipophilic domain which is responsible for its interaction with lipid and presumably anchors the proteoglycan in cellular membranes.

Animals↗

PubMed1995

Exocytosis.

Exocytosis is the fusion of secretory vesicles with the plasma membrane and results in the discharge of vesicle content into the extracellular space and the incorporation of new proteins and lipids into the plasma membrane. Exocytosis can be constitutive (all cells) or regulated (specialized cells such as neurons, endocrine and exocrine cells). Regulated exocytosis is usually, but not always, triggered by an increase in the cytosolic free Ca2+ concentration. In neurons and endocrine cells, a small proportion of regulated secretory vesicles are ready to fuse with the plasma membrane in response to cell stimulation, but the majority are kept in reserve for subsequent stimulation by linkage to a filamentous network of synapsins (in neurons) or actin (in endocrine cells). Regulated exocytosis varies greatly in kinetics and Ca2+ dependency between cell types. It is likely that several different Ca(2+)-binding proteins are involved in regulated exocytosis, with synaptotagmin apparently essential for fast exocytosis at synapses. GTP-binding proteins of both the monomeric and heterotrimeric forms are involved in exocytosis, although their precise role is unclear. Intense current interest focuses on the idea that the molecular mechanism of vesicle docking and fusion is conserved from yeast to mammalian brain. The SNARE hypothesis postulates that vesicle SNAREs (synaptobrevin and homologues) mediate docking by binding to target SNAREs (syntaxin/SNAP-25 and homologues), whereupon SNAPs and NSF bind to elicit membrane fusion.

Animals↗

PubMed1992

Retinal degeneration in the pcd/pcd mutant mouse: accumulation of spherules in the interphotoreceptor space.

The Purkinje cell degeneration (pcd) mutant mouse rapidly loses cerebellar Purkinje cells and about 50% of its retinal photoreceptor cells at between 3 and 5 weeks of age, and thereafter slowly loses the remaining photoreceptor cells during the first year of life. An ultrastructural study of the developing photoreceptor cells of the pcd/pcd retina was undertaken using both transmission and scanning electron microscopy to characterize further the previously reported retinal vesicles associated with this mutation. Transmission electron microscopy (TEM) revealed an abundance of 'bead-like' vesicles or excrescences in the extracellular matrix surrounding the inner segment region at post-natal day (P) 25. The vesicles are membrane bound, amorphous in appearance and vary in size from 125 to 370 nm. Scanning electron microscopy suggests that the vesicles seen with TEM are actually spherules formed from outpocketing and pinching off of the plasma membrane in the mitochondria-rich region of the rod inner segment. At P25, the spherules are concentrated in the interphotoreceptor space at the level of rod inner segments; at P40, however, they are displaced from their origin and appear mostly at the level of rod outer segments and in the subretinal space.

Animals↗

PubMed1988

Ultrastructural localisation of alkaline phosphatase activity in osteoarthritic human articular cartilage.

The distribution of alkaline phosphatase activity in human articular cartilage from normal and osteoarthritic joints has been examined by an electron microscope technique, probably for the first time. In osteoarthritic cartilage chondrocytes and matrix vesicles close to the tidemark were positive for alkaline phosphatase activity. Large numbers of matrix vesicles were found within the extracellular matrix of osteoarthritic cartilage, and there is a specific relation between phosphatase activity, matrix vesicles, and initial mineral formation in the tidemark region of articular cartilage.

Aged↗

PubMed1979

Preparation of subcellular fractions from granulation tissue by density gradient centrifugation.

The 7000 g pellet of homogenized mature sponge-induced granulation tissue was fractionated by centrifugation in a stepwise sucrose gradient in order to study the synthesis and secretion of collagen and other components of this tissue on the subcellular level. As indicated by chemical and enzymatic assays, by electron microscopy and by incorporation experiments, the collagen-synthethesizing rough endoplasmic reticulum fraction was isolated free from the secretory vesicles (smooth endoplasmic membranes and Golgi elements) and fibrous extracellular matrix. Collagen differed from other proteins in the distribution among the subcellular fractions. In pulse-chase experiment the translocation of 14C-labelled collagen was demonstrated from the rough endoplasmic reticulum through the secretory vesicles to extracellular fibrillar collagen. This fractionation method will be used to study the modulation of collagen synthesis and secretion in the reparative tissue.

Animals↗

PubMed2003

The roles of annexins and alkaline phosphatase in mineralization process.

In this review the roles of specific proteins during the first step of mineralization and nucleation are discussed. Mineralization is initiated inside the extracellular organelles-matrix vesicles (MVs). MVs, containing relatively high concentrations of Ca2+ and inorganic phosphate (Pi), create an optimal environment to induce the formation of hydroxyapatite (HA). Special attention is given to two families of proteins present in MVs, annexins (AnxAs) and tissue-nonspecific alkaline phosphatases (TNAPs). Both families participate in the formation of HA crystals. AnxAs are Ca2+ - and lipid-binding proteins, which are involved in Ca2+ homeostasis in bone cells and in extracellular MVs. AnxAs form calcium ion channels within the membrane of MVs. Although the mechanisms of ion channel formation by AnxAs are not well understood, evidence is provided that acidic pH or GTP contribute to this process. Furthermore, low molecular mass ligands, as vitamin A derivatives, can modulate the activity of MVs by interacting with AnxAs and affecting their expression. AnxAs and other anionic proteins are also involved in the crystal nucleation. The second family of proteins, TNAPs, is associated with Pi homeostasis, and can hydrolyse a variety of phosphate compounds. ATP is released in the extracellular matrix, where it can be hydrolyzed by TNAPs, ATP hydrolases and nucleoside triphosphate (NTP) pyrophosphohydrolases. However, TNAP is probably not responsible for ATP-dependent Ca2+/phosphate complex formation. It can hydrolyse pyrophosphate (PPi), a known inhibitor of HA formation and a byproduct of NTP pyrophosphohydrolases. In this respect, antagonistic activities of TNAPs and NTP pyrophosphohydrolases can regulate the mineralization process.

Alkaline Phosphatase↗

PubMed2006

Mechanisms of peptide hormone secretion.

According to the classical view, peptide hormones are stored in large dense-core vesicles that release all of their cargo rapidly and completely when they fuse with and flatten into the plasma membrane. However, recent imaging studies suggest that this view is too simple. Even after vesicles fuse with the plasma membrane, cells might control the rate of dispersal of vesicle cargo - either by modulating the properties of the fusion pore that connects the vesicle lumen to the extracellular solution or by storing cargo in states that disperse slowly in the extracellular space. Understanding these mechanisms is important, owing to the increasing prevalence of diseases, such as type 2 diabetes mellitus, which arise from insufficient secretion of peptide hormones.

Animals↗

PubMed2001

Imaging synaptic zinc release in living nervous tissue.

Zinc enriched neurons have a pool of synaptic vesicles which contain free or loosely-bound zinc ions. The movement of the vesicular zinc ions into the synaptic clefts has been previously studied by microdialysis, fluorescence postmortem staining for zinc and radioactive zinc isotope. In this study the zinc fluorescence probe N-6-metoxy-p-toluensulfonamide quinoline (TSQ) has been applied as a tracer of synaptic release of zinc ions. This fluorochrome permeates cell membranes and when exposed to living brain slices gives rise to a staining pattern similar to that seen with autometallography. In the living brain slices, fluorescence emission persists after exposure to calcium saturated ethylen diamino-tetra-acetic acid (Ca-EDTA) because this chelator does not penetrate cell membranes, while sodium dethyldithiocarbamate (DEDTC), that does penetrate membranes, partially suppressed the fluorescence emission. Stimulation of slices bathed in the non-permeant chelator Ca-EDTA with 50 mM potassium chloride leads to a rapid and complete disappearance of fluorescence. In the absence of Ca-EDTA, however, potassium stimulation induces a sudden transitory increase in fluorescence. This increase is caused by a translocation of the fluorochrome (TSQ) zinc molecules from the weakly acid interior of the synaptic vesicles to the neutral extracellular space, whereby the fluorescence emission of the molecules is enhanced sufficiently to be recorded by a high sensitivity TV camera.

Aminoquinolines↗

PubMed1987

Ultrastructural localization of type IV collagen, laminin and prolyl hydroxylase in biliary epithelial cells of rat liver following ligation of the common bile duct.

Type IV collagen and laminin are major components of basement membrane (BM), whereas prolyl hydroxylase (PH) is a key enzyme in the hydroxylation of proline to hydroxyproline in collagen synthesis. In order to elucidate the exact mechanism of the formation of BM, immune electron microscopic observation of type IV collagen, laminin and PH was made in rat liver with marked proliferation of bile ducts following ligation of the common bile duct. Extracellular localization of type IV collagen was found in the BM of bile ducts and blood vessels and in the space of Disse in both normal rat liver and the liver of rats undergoing operation. Type IV collagen was localized in lamina rara and lamina densa. Laminin was codistributed with type IV collagen in BM but rarely in the space of Disse even in the liver of rats undergoing operation. Immunostaining of laminin was diffusely spread in lamina densa, but sparsely in lamina rara. Though no reaction products of type IV collagen and laminin were detected in the cytoplasm of normal biliary epithelial cells, they were found in rough endoplasmic reticulum (rER) and the vesicles close to the basal surfaces of the plasma membrane of the proliferating biliary epithelial cells. No evident localization of these components in Golgi apparatus was found. PH was found in rER of the biliary epithelial cells, hepatocytes, endothelial cells of vessels, fibroblasts and perisinusoidal cells except for Kupffer cells in normal rat liver. More intense and diffuse staining of PH was observed in rER in the proliferating biliary epithelial cells of the liver of rats undergoing operation in concomitance with the evident localization of type IV collagen in this organelle. These findings suggest that the major components of BM, such as type IV collagen and laminin in the proliferating biliary epithelial cells, are produced in rER and secreted by vesicles to the basal extracellular spaces, thus forming new BM in these circumstances.

Animals↗

PubMed2006

Osteoblast behaviour on in situ photopolymerizable three-dimensional scaffolds based on D, L-lactide, epsilon-caprolactone and trimethylene carbonate.

Polymer networks formed by photocrosslinking of multifunctional oligomers have great potential as injectable and in situ forming materials for bone tissue engineering. Porous scaffolds varying in polyester type and crosslinking density were prepared from methacrylate-endcapped oligomers based on D,L-lactide, epsilon -caprolactone and trimethylene carbonate: LA/CL-hexanediol, LA/CL-dipentaerythritol and LA/TMC-HXD. The biocompatibility and bone formation were related with the degradation time and mechanical properties. The viability of fibroblasts was evaluated after incubation with extraction medium by MTT-assay. All scaffolds showed a good biocompatibility. Rat bone marrow cells were cultured on the scaffolds for 21 days and were able to attach and differentiate on the scaffolds. The cells expressed high alkaline phosphatase activity, have formed a mineralized extracellular matrix and secreted osteocalcin. TEM of the polymer interface revealed osteoblasts which secreted an extracellular matrix containing matrix vesicles loaded with apatite crystals.LA/TMC-HXD, LA/CL-HXD and LA/CL-DPENT had a 50% mass loss at 3,5 months respectively 6 and 7, 5 months. The mechanical properties improve by increasing the branching of the precursor methacrylates (by replacing HXD by DPENT) but do not depend on their chemical composition. Hence, scaffolds with high elastic properties and variable degradation time can be obtained, which are promising for bone tissue engineering.

Alkaline Phosphatase↗

PubMed1993

Response of bone and cartilage cells to biomaterials in vivo and in vitro.

In vivo and in vitro models have been developed to study the bone/material interface. The in vivo model exploits the osteogenesis that accompanies marrow ablation of the rat tibia and uses morphological and biochemical changes in extracellular organelles, called matrix vesicles, as markers of the healing process. Matrix vesicles, which are associated with primary bone formation and calcification, are produced by osteoblasts and are sensitive to cellular and environmental regulation. In bone adjacent to bone-bonding implants, matrix vesicle number increases, as does its alkaline phosphatase activity. In bone adjacent to nonbonding materials, matrix vesicle activity is inhibited. The materials exert systemic effects which can also be studied by use of matrix vesicles. Cell models are needed in order for the specificity of the cellular response to the material to be understood. By the use of culture plates sputter-coated with implant materials, the response of cells can be studied under controlled conditions. Comparison of the response of costochondral chondrocytes at two stages of endochondral development demonstrates that the effects of various materials are surface- and cell-maturation-dependent. Cells cultured on Ti exhibited increased alkaline-phosphatase-specific activity, whereas those cultured on Al2O3 have decreased enzyme activity.

Alkaline Phosphatase↗

PubMedDate not supplied

Tympanosclerosis. An electron microscopic study of matrix vesicles.

The ultrastructure of tympanosclerotic tissue has been studied and compared with other processes of fibrogenesis and calcification. Evidence is presented of the presence of large numbers of extracellular membrane-bound matrix vesicles and their role as the site of primary calcification in tympanosclerosis. Calcified spherular structures are formed and fuse into larger structures lined by a lamina limitans indicating the lines of arrested calcification in the collagenous matrix.

Adult↗

PubMed2005

Bacterial outer membrane vesicles and the host-pathogen interaction.

Extracellular secretion of products is the major mechanism by which Gram-negative pathogens communicate with and intoxicate host cells. Vesicles released from the envelope of growing bacteria serve as secretory vehicles for proteins and lipids of Gram-negative bacteria. Vesicle production occurs in infected tissues and is influenced by environmental factors. Vesicles play roles in establishing a colonization niche, carrying and transmitting virulence factors into host cells, and modulating host defense and response. Vesicle-mediated toxin delivery is a potent virulence mechanism exhibited by diverse Gram-negative pathogens. The biochemical and functional properties of pathogen-derived vesicles reveal their potential to critically impact disease.

Animals↗

PubMed2000

Primary mineralization at the surfaces of implants.

Osteogenesis around implants is affected by the physical and chemical characteristics of the biomaterials used. The osteoprogenitor cells must migrate to the implant site and synthesize and secrete a mineralizable extracellular matrix. Because this is neo-bone formation, the mechanism by which the cells calcify their matrix involves extracellular organelles called matrix vesicles in a process termed "primary mineralization". Two different methods for assessing the effects of implant materials on primary mineralization are presented in this report. In the first approach, different implant materials used in dentistry and orthopedic surgery were placed in rat tibial bones after marrow ablation. Two groups of implants were used, bone-bonding and non-bonding materials. We examined the effects of the materials on calcification morphometrically by quantitating changes in matrix vesicle morphology and distribution in endosteal tissue around implants as compared with normal endosteal bone healing. In addition, matrix vesicles were isolated from the endosteal tissue around the implant as well as from the contralateral limb and were examined biochemically. The results demonstrated that bone-bonding materials induced a greater increase in matrix vesicle enzyme activity than did non-bonding materials. However, all materials caused changes in matrix vesicles that were different from those seen in normal endosteal bone formation following injury. The effects of implant materials on biochemical markers of mineralization, including specific activities of matrix vesicle alkaline phosphatase and phospholipase A2 and phosphatidylserine content, demonstrated a high correlation with the morphometric observations with regard to enhancement and/or delay of primary mineralization. In the other approach, we used a radioisotopic method to evaluate the effects of implant materials on primary mineralization. This analysis revealed that implants alter bone healing, as shown by the differential uptake of 99mTc and 32P in different bone compartments. Decreased 32P uptake by the organic phase in the presence of bone-bonding implants suggests that cleavage of 99mTcMD32P into its technetium and methylene diphosphonate moieties was inhibited by the presence of the implants. In summary, these approaches to evaluating the effects of materials on primary mineralization demonstrate that the marrow ablation model can easily distinguish between bone-bonding and non-bonding materials. The use of this model can be valuable in the development of new materials.

Animals↗

PubMed1979

Morphological studies of stimulated adrenergic axon varicosities in the mouse vas deferens.

The postganglionic axons of sympathetic neurons innervating the mouse vas deferens were stimulated transmurally in vitro by passing square pulses between two platinum electrodes. The ultrastructural appearance of the adrenergic nerve terminals was compared to samples fixed immediately after 30 min of stimulation and in samples allowed to recover for 2 h before fixation. The contralateral vasa deferentia served as controls, and these were incubated in Krebs solution for the same period as stimulated muscles. For each of four experiments, the mean number of large and small dense-core vesicles per square micrometer was calculated, as were the mean area and perimeter of the axon varicosities in each group. It was found that the number of small vesicles per square micrometer decreased by 60% during the stimulation period, but returned almost to control levels 2 h later. Large vesicles did not change in number during the stimulation or recovery periods. The proportion of vesicles containing cores was also determined for each group and found to decline just after stimulation in the small vesicle population, but to remain constant in the large vesicle population. The core depletion was partly reversed after 2 h. The vesicle recovery process was studied by use of the extracellular tracer horseradish peroxidase (HRP). When HRP was present in the extracellular space during stimulation, large numbers of vesicles contained the marker after recovery from stimulation. Thus, it is proposed that adrenergic axon varicosities recycle vesicle membrane through the plasma membrane in a manner similar to that already described for cholinergic nerve terminals.

Animals↗